Peptide compounds



answer PEPTIDE MPUUNDS Urs Gloor, Riehen, lleter Quitt, Basel, Rolf Strider, Neuallschwil, and Karl Vogler, Riehen, Switzerland, assignors to Hofimann-La Roche line, Nutley, N..ll., a corporation of New Jersey No Drawing. Filed May 29, 1962, Ser. No. 198,455 Claims priority, application Switzerland, June 1, 1961, 6,366/ 61; Dec. 1, 1961, 13,988/61 15 Claims. (Cl. 26illl2.5)

This invention relates to novel peptide derivatives, their use, and intermediates in their manufacture. More particularly, the novel peptide compounds of the invention are selected from the group consisting of compounds of the formula.-

and acid addition salts thereof; wherein m and n are each a whole integer from 0 to l; X is selected from the group consisting of hydroxy, alkoxy, amino, alkylamino, dialkylamino, and hydrazine; each R is the residue of an as amino acid devoid of its a-amino and carboxyl group, at least one R being the residue of adiaminocarboxylic acid; and the molecule contains at least one long-chain aliphatic group selected from the-group consisting of a long-chain aliphatic acyl substituent on an amino acid-component amino group not partaking in a peptide linkage and a longchain aliphatic substituent comprehended by X when X is selected from the group consisting of alkylamino and dialkylamino.

The compounds of Formula I above are dipcptides, tripeptides, or tetrapeptides, derived from a-arnino carboxylic acids such as sen'ne, phenylalanine, tyrosine, leucine, lysine, argininc, ornithine, a,'y-diarninobutyric acid and a,fldiaminopropionic acid, and the like, linked in u-amidc fashion. Especially preferred as building component ot-amino carboxylic acids are diamino-carboxylic acids such as lysine and the basic acids named thereafter in the foregoing exemplary listing. Thus, where R in Formula I above is the residue of a diaminocar-boxylic acid it can be represented by the formula:

I NH:

wherein R is selected from the group consisting of lower alkylenev (preferably methylene, dimethylene, :trimethylene or tetramethylene) and When R is the latter group, R of course is the residue of arginine. Also preferred as a-amino acid components are optically active amino acids such as L-lysine, the preferred optical form being the L-form. Compounds I'll Patented August 9, 1966 of Formula I are especially preferred wherein n; and 11 are each 0, that is the dipeptide compounds are preferred. One preferred group of compounds of Formula I above are those wherein X is lower alkoxy such as methoxy, eth-oxy or the like and the long-chain aliphatic acyl group contains at least 10 carbon atoms such as a higher fatty acid moiety as palmitoyl, stearoyl, or the like.

Another preferred group of compounds of Formula ll above are those wherein X is alkylamino or dialkyla-mino and at least one of the amido N-alkyl suhstituents is of at least 10 carbon atoms such as cetyl, lauryl, or the like. A narrower preferred embodiment consists of compounds of Formula I wherein the long-chain aliphatic group contains 12 or more carbon atoms. The term aliphatic as used herein includes both straight, e.g. palmitoyl, and branched, e.-g. phytanoyl, chain alkyl moieties, as well as saturated, e.g. pentadecanoyl, and unsaturated, e.g. undecanoyl, alkyl. Preferred long-chain aliphatic groups contain not over 20 carbon atoms. it will be appreciated that in the above denotations of carbon atom content of the long-chain aliphatic moiety, the number of carbon atoms with respect to a slong-chain aliphatic acyl moiety includes the carbon atom partaking in the carbonyl group. For example, palmitoyl contains 16 carbon atoms. Especially preferred long-chain aliphatic groups are those having from 14 to 18 carbon atoms.

In Formula 1 above, the consecutive R-symbols can represent residues derived from the same or different rx-amino carboxylic acids. This will. be apparent from the following exemplary list of preferred compounds: [N-palmitoyl-L-lysyl]-L-lysine, L-lysyL[N -palmitoyl-l; lysine], [hP-palmitoyl-L-(a,'y-diaminobutyryl-L-( org-diaminobutyric acid), L-(a -diaminobutyryl)-L-[N"-pal mitoyLL-(a diaminobutyric acid], (N pa-hnitoyl-L-or" nithyl) -L-tan'ginine (N-pa1mitoyl-L-ornithyl) -L-ornithine (N-palmi-toyl-L-anginyl)-L-arginine and their esters (especially their lower alkyl esters, such as their methyl and ethyl esters), amides and 'hydrazides. The con1- pounds of Formula 1 above form acid addition salts, e.g. non-toxic acid addition salts, with both inorganic and organic acids, such as hydrohalic acids, phosphoric acid, nitric acid, acetic acid, formic acid, and the like.

The compounds of Formula I above and acid addition salts thereof can be prepared in accord with processes which are known per se in peptide chemistry. Thus, for example, they can be built up from theappropriate ocamino carboxylic acids or appropriate protected peptides with the use of condensing agents, or via mixed anhydrides, azides, activated esters, or acid chlorides, the terminal carbonyl function of which can, if desired, be hydrolyzed to the free hydroxyl group or be converted into an amide, N-alkylamid-o, N,N-dialkylamido or hydrazido group. The protecting groups can be removed and the resulting peptide compounds, if desired, converted into their salts and a long-chain acyl group introduced by interchange with a protecting group which can be selectively split oif.

According to one method, compounds of the formula:

H NR-C0{NH-R-CO} {-NHRCO} -Y (II) can be reacted with a compound of the formula:

wherein a. I), c, and (1 are each a Whole illegal from t to l and lhc total of a, b. c,-anci r! is a whole integer from to 2', Y is selected from the group consisting of halogen, hydroxy, nzidc, phcnoxy, nita'ophenoxy, phenylmercapto and the residue of an anhydride with an inorganic or organic acid; X is selected from the group consisting of alkoxy, amino, alkylamino, dialltylamino, and hydroxy in the form of a salt with an inorganic or tertiary organic base, eachR has the same meaning as in Formula I above; and all amino groups, with the exception of the one terminating the chain of Formula III, are protected by removable protecting groups and/01' long-chain acyl groups.

The reaction between the compounds of Formulae II and HI above comprises a condensation together with a splitting off of the compound HY so as to form a dipeptide, tripeptide, or tetrapeptide of the formula:

wherein n 11 each R and X have the same meaning as above, and in which all amino groups are protected in the manner indicated with respect to Formulae l1 and III above.

Those peptides of Formula IV which neither contain a long-chain N-a-cyl group nor in the minal COX group a long-chain alkyl substituent are then converted into corresponding peptides substituted by long-chain N-acyl or N-alkyl groups by interchange of at least one selectively removable protecting group for a long-chain acyl group, or by introduction of a long-chain N-alkyl substituent into the terminal carboxyl function. The removable protecting groups can be split off from the resulting longchain aliphatic substituted peptide and, in the case of the long-chain acyl substituted peptide, the peptide product, prior to the splitting olf, can, if necessary, be converted into its hydrazide. Finally, if desired, the peptide products can be converted into their acid addition salts.

As usual in peptide chemistry, the reaction groups which do not take part in the formation of the peptide are protected prior to the condensation reaction involving the splitting oil of HY. Such protection is readily accomplished in the case of the carboxyl group by conversion to a corresponding ester or amide or by salt formation with an inorganic base or an organic tertiary base. Free amino groups can, for example, be protected by conversion into benzyloxycarbonylamino groups (i.e. their corresponding carbobenzoxy groups) for example, by the action of benzyloxycarbonyl chloride in a Shotten-Baumann reaction. The carbobenzoxy protecting groups can subsequently be split oil by catalytic hydrogenation. Also, as far as the condensation is concerned, the longchain N-acyl groups can themselves serve as protecting groups. The amino groups of the intermediate amino acids or peptides can also be protected via their conversion into phthalyl derivatives; for example, by heating the amino acid or peptide with phthalic acid anhydride. In this embodiment, the phthalylprotecting groups can be removed from the protected amino group after the condensation via the addition of hydrazine and treatment with hydrochloric acid. The amino groups can also be protected by formylation by reacting the intermediate amino acids or peptides with formic acid in the presence of acetic anhydride.

By having one or more of the amino groups protected by a formyl protecting group and one or more amino groups protected by a carbobenzoxy group, it is possible to selectively remove one sort of the protecting groups, since only the carbobenzoxy groups are removable by catalytic hydrogenation or by the action of a hydrobromic acid/acetic acid mixture while only the formyl groups are removed by the action of mineral acids in the cold.

it is thus possible to introduw the long-chain'Nacyl substiluent immediately after the condensation. Such in l'IHtlLlCtltlll can, for example, be effected via the action of an appropriate acid chloride such as, for example, palmitoyl chloride.

The following synopses are illustrative of the methods available for preparing the compounds of this invention:

CARBODIIMIDE METHOD This method is applicable when there is used as a starting material a compound of Formula Ill above in which Y is hydroxy, i.e. an amino acid, dipeptide or tripeptide bearing a terminal free carboxylic group. The method comprises effecting the condensation of a starting material of Formula ll wherein Y is hydroxy with a starting material of Formula Ill in the presence of a condensing agent, such as a carbodiimide, cg. dicyclohexylcarbodiimide, or carbonyldiimidazole. The condensation is ad vantageously carried out in a solvent at low temperature. Suitable solvents are organic solvents such as chloroform, N,N-dimethylformamide, ethyl acetate, or the like. The urea formed from the condensation agent can be removed by filtration, and the peptide product, which remains in solution, can be isolated from the filtrate.

AZIDE METHOD ACID CHLORIDE METHOD This method comprehends formation of the peptide by reaction of an acid chloride group (Y=Cl) with an amino group. One can conveniently couple a carbobenzoxy amino acid chloride (obtained via the action of thionyl chloride) with an amino acid ester (X =a-lkoxy) in cold in the presence of a base.

METHOD UTILIZING MIXED ANHYDRTDES This method comprehends using for the condensation a compound having as its carboxyl function (i.e. -COY) a mixed anhydride residue formed with an organic or inorganicacid. Suitably, a carboxyl compound of Formula 11 above (Y=OH) is treated in an inert solvent (tetrahydrofuran) with a base, for example, tricthylamine, the resulting salt reacted with a chlorocarbonic acid ester at a low temperatune, and the resulting mixed anhydride reacted without isolation with an amino acid ester (X =alkoxy) or a sodium salt or" Formula Ill above (X =ONa). Exemplary of the chlorocarbonic acid esters which can be employed, are the lower alkyl esters such as the methyl, ethyl or t-buty1 esters of chlorocarbonic acid. The active anhydride can also be prepared using sulphur trioxide in dimethylformamide, diethylphosphorochloridite (C H O P-Cl] tetraethyl- 'pyrophosphate [(C H O) POP(OC H or the like.

METHOD UTILIZING ACTIVE ESTER This method comprehends using as a starting material of Formula 11 above a compound in which the carboxyl function (-COY) is a so-called active ester group such as, for example, cOY p-nitrophenyl ester. Such an active ester can be obtained from a protected amino acid and di(p-nitrophenyl)-sulphite in the presence of pyridine and, on reaction at room temperature with an amino acid ester of Formula Hi (X =alkoxy) gives the desired peptide compound which can then he worked up by crystallization. Exemplary of suitable active ester groups are the p-nitrophenyl, 3,5-dinitrophenyl, thiophenyl, and the like ester groups.

Intermediate compounds of the formula:

wherein R has the same meaning as above; Y is selected from the group consisting of alkoxy (preferably lower alkoxy) and hydroxy; and at least one of the two amino groups is substituted by a long-chain acyl group of at least carbon atoms and the other is protected with a removable protecting group, are novel compounds and form a part of this invention. Preferred compounds of the formula:

described above are N-palmitoyl-I-l"-benzyloxycarbonylornithine, N"-palmitoyl-N -benzyloylcarbonyl L-lysine, N palmitoyl N benzyioxycarbonyl-D-lysine, N"-palmitoyl N-benzyloxycarbonyl-DL-lysine, N"-stearoyl-N- benzyloxycarbonyl-L-lysine, N-lauroyl-N -benzyloxycarbonyl-L-lysine, N"-pa1rnitoyl-Nbenzyloxycarbonyl-L-an, diaminobutyric acid, N-capryl-N-benzyloxycarbonyl-L- lysine and N"-(10-undecenoyl)-(N-phthaloyl)-L-lysine. I

Also within the scope of the invention are novel intermediates of the formula:

wherein at least one bf the two amino groups is substituted by a long-chain acyl group of at least 10 carbon atoms; R has the same meaning as above; and Y is selected from the group consisting of alkoxy (preferably lower alkoxy) and hydroxy. Exemplary of such compounds are N-palmitoyl-L-lysine and its lower alkyl esters and N'-polmitoy1-L-(a -dianiinobutyric acid) and its lower alkyl esters.

Compounds of Formula I above and their non-toxic acid addition salts are useful as preserving and disinfecting agents. They are active against gram positive and gram negative pathogenic and apathogenic bacteria, fungi, and yeasts such as Escherichia coli, Pseudomonas aeruginosa, Proteus vulgarz's, Staphylococcu aureus, Bacillus cereus, Bacillus subtilis, Streptococcus fecalis, Lactobacib Ius plantarum, L. acidolplzflus, L. buchnerii, L. brevis, Stzccharomyces fragilis, S. cerevisiae, Candida gztz'llicrmodii, Pichia farinosa and Pennicillurn sp. spores. These activities, in part, are illustrated by the exemplary practical applications given in the working'examples below. The compounds decompose under the influence of proteolytic enzymes to give essential amino acids or very closely related compounds which no longer have any antibacterial activity. The compounds of Formula I thus do not give rise to any residue problem and are of low toxicity. Also, their good water-solubility facilitates their use. Compounds of Formula I above are suitable for preserving both solid and liquid foods. Thus, another embodiment of this invention comprehends a food composition which comprises an edible material treated with an amount b suiiicicnt to effectively preserve said edible material of a peptide compound selected from the group consisting of compounds of the formula: 1

and non-toxic acid addition salts thereof; wherein n 11 each R and X have the same meaning as above. In one preferred aspect of the invention, the peptide compound used is comprised of one or more L-arnino acid components. It is further preferred that the long-chain acyl or alkyl group should contain at least 10, preferably from 10 to 20, carbon atoms such as, for example, the palrnitoyl or stearoyl radical. Still more preferred are those containing from 12 to 20 carbon atoms, and especially preferred are those containing from l4-18 carbon atoms. Exemplary of peptide compounds embodying these preferred aspects of the invention are N -palmitoyl-L-lysyl-L-lysine, L-lysyl-N palmitoyl L-lysine, [N-pa1mitoyl-L-(a,y-diaminobutyryl) ]-L-(ot,'y diaminobutyryl)-L-[N"-palmitoyl]L(a,'y-diarnin obutyric acid), N pa-lm-itoy-l-L-ornithyl]-L-arginine, [N-palmitoyl L-ornithyl] L-ornithine, [N-palmitoyl-L-orginyl]-L-arginine and esters, amides and hydrazides thereof.

It will be appreciated from the foregoing exemplary listing that diarnino carboxylic acid as used herein comprehends arginine, as well as lysine and the like. in a fur ther preferred aspect of the invention, the peptide compound used as a preservative is a dipeptide. The edible materials which may be preserved by a treatment with the peptide compounds of this invention are exemplified by both solid and liquid foods, for example, meat, meat products, fish, fish pastes, fish preserves (including those of lower pH values), sea-food (shrimps, etc), poultry, fresh vegetables, fresh fruit (e.g. resisting the infection ofcitrus fruits by penecillium moulds), fruit juices, bread and bakery products and cheese, as well as animal feeds of different types. The peptide compounds of Formula I above and acid addition salts thereof also are useful as emulsifying agents.

Another aspect of the invention comprehens a process for preserving edible material which comprises -treating said edible material with an amount suflicient to preserve said edible material of a peptide compound of For mula 1 above, or a non-toxic acid addition salt thereof. The word treating is used above in its generic sense and comprehends admixing, coating, impregnating, contacting, and the like; for example in the case of a liquid food, a composition containing the preservative peptide compound can merely be admixed with the food. in the case of a solid food, treatment can be effected, for example, by spraying the food with a solution containing the peptide compound, or by soaking the food in a solution containing the peptide compound. These methods are illustrative of direct methods of treatment. Indirect methods of treatment can also be used, for example, the food to be preserved can be packaged in a wrapping material which carrice the preservative material. For example, the wrapping material can be coated or impregnated with the preserva tive material. The peptide'preservative agents can be added as such (e.g. in powder form) or in the form of an aqueous solution, an emulsion or dispersion, or in the form of a paste or gel, to the materials to be preserved, intimately mixed therewith, and the goods to be preserved suitably sealed or packed. In certain cases, the preservative agents can be added to the wash-water (such as in preserving potatoes, onions, bulbs, strawberries). They can also be introduced into ice which is intended for keeping fish and other sea-food cold during storage. The pre serving agents of this invention may be used in combination with one another or in combination with other preserving measures. Thus, by use of the new agents in association with a heat sterilization process, the duration of heating is lessened. Also, the agents can be used in association with low temperature storage. For-example, for

refrigerated transports, such as the transportation of citrus fruits.

'In the examples, the following abbreviations are used:

Palm

DMF

Arg

Nitroarg.

Dab

Lys

Ser

Leu

lhc;

Tyr

llcnzyloxycurhonyl Pulmltoyl Diructhyll'ormamlde Arglnyl Nitroarginyl a,y-Diaminobutyryl Lysyl Ornithyl Phenylalanyl Tyrosylu Ucmocol CH1;

w (an:

I OH

As illustrative of the above usage in the following examples there can be cited, for example, N-palrnttoyl-(l\lcarbobenzoxy -L-lysyl- N-carbobenzoxy) -]Llysine methyl ester of the formula:

l JO OCHzCsH5 C O CHnCoHs h which in the following examples is shown as H-L-NitroargOC H 1 The following examples are illustrative but not limitative of the invention. All temperatures are in degrees ccntigrade.

Example suction, washed with water and dried in vacuo at 70. After recrystallization from ethyl acetate/petroleum ether N-Palm-L-Lys-L-Lys-OCH -2HC1; 50 g. of N-Palm- (N-Z)-L-Lys-(N-Z)-L-Lys-OCH was dissolved in 50 ml. of glacial acetic acid with warming, and shaken with the exclusion of moisture for 1 /2 'hours at 20 with ml. of 33% hydrobromic acid/glacial acetic acid. The mixture was then degasified for 15 minutes under a waterjet vacuum, diluted with 150 ml. of water and twice extracted with ether. The aqueous phase was then, with the addition of ice, made alkaline with concentrated urn-- monia and extracted several times with ethyl acetate. The ethyl acetate extracts were dried over sodium sulfate and concentrated in vacuo at 40. The residual syrup was dissolved in .20 ml. of methanol and adjusted to pH 7 with 4 N methanolic hydrochloric acid and the so-formed neutral solution evaporated in vacuo at 40. The soobtained residue was then crystallized from acetone yielding N-PalmL-Lys-L-Lys-OCH 'ZHC] melting at; 210- 212 (doc); [a] =-l.7 (0:2 in water).

N-Palm-(N-Z)-L-Lys-(N-Z)-L-Lys-OH: 26 g. of 1 Palm-(N -Z)-L-Lys-(NZ)-L-Lys-OCH was dissolved in 1 liter of methanol and saponifiecl for 16 hours at 20 with 50 ml. of 2 N sodium hydroxide. The reaction mixture was then filtered and the filtrate concentrated in vacuo at 35 to a volume of 100 ml., mixed with 1 liter of 0.01 N hydrochloric acid, filtered under suction and the precipitate washed with water and dried in vacuo at 70. The sci-obtained residue was crystallized from ethyl acetate/petroleum ether yielding N-Palm-(N-Z)- lo-Lys-(N lhblys-OH melting at 129-431".

N-Palm-L-Lys-L-Lys-OH.HCl: 16 g. of N"-Palm-(N- Z)-L-Lys-(N-Z)-L-Lys-OH in 300 ml. of glacial acetic acid and 30 ml. of water was hydrogenated following the addition of l g. of palladium activated charcoal. The catalyst was then filtered off with suction, the filtrate concentrated in vacuo at 45 and the residue twice subjected to dissolution and solvent evaporation with methanol. The so-obtained residue was then taken up in a small amount of water, the pH of the so-obtained solution adjusted to 7 with 1 N hydrochloric acid, the soohtained neutral solution filtered, mixed with acetone and again filtered, yielding, as the precipitate, N-Palm-L-Lys-L- Lys-OHHCl which decomposed at temperatures above [a] =-4- (c.=2 in water).

Example 2 N-Palm-(N-Z)-L-Lys-(N-Z)-L-Lys-NH 20 g. of N-Palm-(N-Z)-L-Lys-(N-Z)-L-Lys-OCI-I in 800 ml. of methanol was saturated with ammonia gas at 25 and permitted to stand at 25 for 48 hours. The precipitate was filtered 01? with suction, washed with a large volume of water, dried in vacuo at 60 and crystallized from dirnethylformamide/Water yielding N-Palm-(l l- Z) L Lys-(N-Z)-L-Lys-NH melting at 174-l'76; [a]; =--8.2 (c.=2 in 'dimethylformamide).

. 9 l@ N-Palm-L-Lys-L-Lys-NH .2HCl: 17 g. of N-Palm- Me=methanol (N-Z)-L-Lys-(N-Z)-L-Lys-NH in 300 ml. of glacial Et=ethanol acetic acid and 30 ml. of water was hydrogenated in Aq=water the presence of 1.7 g. of palladium activated charcoal. Ac=acetone The catalyst was then filtered off with suction and the E=ethyl acetate 5 filtrate concentrated in vacuo at 45. After twice dis- P petroleum ether solving the residue in water followed by subsequent DMF=dimethylformatnide evaporation, the resulting residue was again dissolved in F=melting point a small amount of water and the so-obtained solution Aet=ether F: C. lulu a N -Palm-(N -ZyL-Lys-(N -Z)- -Lys-OC:H 110-113(Et/Aq) N Palm-L-Lys-L-Lys-O(72115.2HC1 242-246(Et) -23(e=3,Aq). b N -PaIImL-LYS-LLYS-OCzH5.2IIaPO4 165(A0/Aq) -20(e=3,Aq) c N -Palrn-(N -Z)-D-Lys-(N -ZyD-LyS-OC H 110-113(Et) N -PaIm-D-LyS-D-LyS-OC2H52HC1 242-246(Et) +23(c=3,Aq).

N -Palm-(N D-DL-Lys-(N-Z)-DL-Lys-OO H 9340mm N -ralmmL-L su)L-L s-ocnitzflor 210-220 (Et) e N -Stearoyl-(N -Z)-L-Lys-(N=-Z)-L Lys-G;H 105-108 (Et/Aq)- N -Stearoyl-L-Lys-L-Lys-OCzH5.2HCl 235-240 Et). -21- (e=3, Aq). r N Palm-(N -Z)-L-Orn-(N -Z)-L-0rn-OCH 121-124 12 -1 9(c=1,DMF)

N -PaIm-L-Orn-L-Oru-OOHMHGI 230-233(Me/Ac) 12.2(c=2,Me). g N -PaIm-(N -DL-Orn-L-Nitroarg-OCH; 135-139(11 -2 5(c=1,DMF),

N -Pahu-L-Orn-UArg-OCI-IaZHC1 217-220(Me/Ac) -5.2 (c=2, Me). n N-Lauroyl-(N=-Z)-L-Lys-(N=-Z)-L-Lys-O CH3 131-139 (Me/Ac)" 11.6 (=2, Me) N -Lauroyl L-LysL-Lys-(JCH3.2HC1. 212-214(Mc/Ae) -20.0(e=2,Mo) 1 s. N PaIm-(Nv-Z)-L-Dab-(Nv-Z)-L- Dab-OCH3 119121(E/P) N -Palm-(Nv-Z)-L-Dab-(N -Z)-L-Dab-NHNH;| 183-184(DMF/Ac) -13.8(e=2,DMF) N -Pnlm-L-Dab-L-Dab-NHNHMHCI 184(Me/Et) N ,N-Di-Z-L-Lys-(N PaImyL-LyS-OCH 151153(Me/Aq) H-L-Lys-(N -Palin)-L-Lys-OCH3.2HC1 170 (Me/Et) 1 N,N-Di-Z-L-Lys-(N=-Paln1)-L-Lys-NHNH 178-180 (Aq H-L-Lys-(N Palrm-LLys-NHNHz.HB1 lat-s (Et Aet) n1 N -Capryl-(N Z)-L-Lys-(N=-Z)-L-Lys-00H 130-132(Me/Aet) -11.5(c=2,Me) N -Capryl-L-Lys-L-Lys-OGHaJZl'ICl 206-211(Me/Aet) 19.9(e=2,Me). n N -Palm-(N Z5-L-Lys-L-ser-oCH3 '125127(Me) N -PaLm-L-LyaL-Ser-OCHMHCI 139 (Aq/Ac) o N -Palm-(N -Z)-L-Lys-L- Lcu-0CHi seas (E) c N Palm-L-Lys-L-Leu-O(.llIa 181-185(Aq) c Nwarm (N -ZrL-LysL-Tyr-0CH3.-. 105-108(E) -1 =2,DMF) N Pnlm-L-Lys-L-Tyr-O(JHz 159-161(Me/E) -42.e c=1,1r

adjusted to a pH of 7 with 3 N hydrochloric acid and the neutral solution then mixed with acetone yielding as the precipitate, N-Palm'L-Lys-L-Lys-NH .ZHCi melting at 232-233" (dec); [a] -=11 (c.=2 in water).

Example 3 N"-Palm-(N-Z)-L-Lys-(N-Z)-L-Lys-NHNH 26 g. of N-Palm-(N-Z)-L-Lys-(N-Z) -L-Lys-OCH in 400 ml. of methanol was treated with 26 ml. of 100% hydrazine hydrate and heated on the steam bath for minutes. After standing for a further 24 hours,'it was mixed with 800 ml. of Water and filtered with suction. The precipitate was washed with a large amount of Water and dried. Upon crystallization from dimethylformamide/ Example 4 According to the above described procedures the dipeptides listed in the following table were prepared. In the table the following abbreviations are used.

Example 5 N-Z-L-Nitroarg-L-Nitroarg-OC H L-Nitroarg-OH [Z. physiol. Chem. 224, 40 (1934)] was dissolved in 4 00 ml. of absolute tetrahydrofuran and with stirring at -l0 treated with 16.2 g. of earbonyh diimidazole. After being stirred for a further 40 minutes a solution of H-L-Nitroarg-OC H (prepared from about 0.2 mole of the oily hydrochloride with the aid of triethylamine) in ml. of dimethylformamide was added thereto, and the mixture then stirred for a further 4 hours at 0". After evaporation of the solvent in vacuo,

the residue was taken up in 1 N hydrochloric acid whereupon an oil separated out which was triturated several times with water whereby it finally solidified. Upon recrystallization from ethanol/Water, it yielded N-ZL- Nitroarg L Nitroarg OC H melting at 123-125"; [a] =-7.8 (c.=l.0 in alcohol).

N-Palm-L-Nitroarg-L-Nitroarg-OC H 14.6 of N-Z- L-Nltroarg-L-Nitroarg-OC H was decarbobenzoxylated by treatment for one hour with 50 ml. of 25% hydrobromic acid in glacial aEetic acid. The hydrobromide was precipitated in ether, washed several times with ether and twice subjected to dissolution in ethanol followed by subsequent evaporation in vacuo. The residue wasreacted in absolute ethanol with excess triethylamine and evaporated in vacuo. The residue was then taken up in 150 ml. of, absolute pyridine, 4'ml. of triethylamine added thereto and at between l0 and l5 with vigorous stirring, 7.2 g. of palmitoyl chloride was then added thereto dropwise. The mixture was then stirred at 0 for a further 30 minutes, and then the solvents evaporated off in vacuo. The residue was partitioned between ethyl seeaeea 'lll acetate and 3 N hydrochloric acid, Washed rapidly with water and saturated sodium chloride solution, dried over sodium sulfate and concentrated in vacuo yielding N- Palm-L-Nitroarg-L-Nitroarg-OC H melting at 169-173 12 solution was then dried over sodium sulfate and concentrated in vacuo. By this method the following N- acylated dipeptidc esters were obtained. The abbreviations used are the same as thoseused in the table of (following recrystallization from ethanol/ether). 5 Example 4.

b N Myristoyl-(N -Z)-L-LyS-(N'-Z)-L-Lys OCH3 t 128-132 (E/P) ll.1 (c=2,l\lc). N"-Myristoyl-L-Lys-L-Lys-OCHJJHC] 214-240 (Mc/Act) 19.1(c=2,Me).

d..." N -h largaroyl-(N*-Z)-L-T.ys-(N -Z)-L-Lys-OGH 130-132 (Mo/1H1)... 11.8 (c=2,lVle). N Marguroyl-L-Lys-L-LysO(I11321161 N 216420 (Me/Aet) -1fi.5 (c=2,Me).

N -Araehidoyl-(N -Z)-L Lys-(N'-Z)-L-Lys-OCH 120-123 (E/P) N -Aracliidoyl-L-Lys-L-Lys-t)(311321101 220-225 (Me) 16.5 (c=2,Me).

f N -Phytanoyh(N=-Z)-L-Lys'(N -Z)-L-Lys OCI1' 115-118 (E/P) 12.9 (C=2,Me). N=-Phytauoyl-L-Lys-L-Lys-OGHa2HCl 215 219 (Me/Aet) -19.5 (c=2,Me).

N -Palm-L-Arg-L-Arg-OC H .ZHCI: N Palm L- Nitroarg-L-Nitroarg-OC H was dissolved in a 50-fold amount of glacial acetic acid and hydrogenated for 24 hours at room temperature following the addition of water and 5% palladium activated charcoal catalyst. The catalyst was then filtered off, the filtrate concentrated in vacuo and the residue crystallized from methanol yielding N Palm-L-Arg-L-Arg-Oc H .ZHCl melting at 225-230"; [a] =-l3.7 (c.=2 in ethanol).

Example 6 N"-Formyl-(N-Z)-L-Lys-(N-Z)-L-Lys-OCH 26.3 g. of N"-F0rmyl-(N-Z)-L-Lys-OH [J. Amer. Chem. Soc, 82, 3727 (1960)] was dissolved in 150 ml. of absolute tetrahydrofuran and treated with stirring at -10 with 13.8 g. of carbonyl-diimidazole. After 30 minutes a solution of H-(N -Z)-L-Lys-OCH (obtained from 28 g. of the hydrochloride by treatment with triethylamine) in 50 ml. of tetrahydrofuran was added thereto, and the mixture then stirred for 4 hours at room temperature. It was then concentrated in vacuo, taken up in ethyl acetate and washed with ice cold 1 M tartaric acid, ice Water, 10% potassium bicarbonate and saturated sodium chloride solution. At this point, sutficient tetrahydrofuran was added to dissolve any precipitates which appeared. if no precipitate appeared, the addition of tetrahydrofuran was omitted. The solution was then dried over sodium sulfate and concentrated in vacuo. The residue was crystallized from acetone/eth :r yielding N"-Formyl- (Nf-Z)-L-Lys-(N-Z)-L-Lys-OCH melting at 147149; [a] =16.9 (c.=1.0 in methanol).

. Deformylation and reaction with fatty acid chlorides: 29 g. of N-Forrnyl-(N-Z)-L-Lys-(NZ)-L-Lys-OCH was shaken for 5 hours with 100 ml. of 2 N methanolic hydrochloric acid and 70 ml. of methanol, yielding a clear solution which was then evaporated in vacuo. The residue Was triturated several times with ether and was twice subjected to dissolution and subsequent evaporation in vacuo, each time with 100 ml. of toluene. The residue was then taken up with 300 ml. of absolute tetrahydrofuran and While cooling with ice treated with 10 ml. of tricthylamine, then filtered and after addition with good stirring of a further 10 ml. of triethylamine, treated with 50 mmole of a higher fatty acid chloride in such a manner so that the temperature did not exceed -10. The reaction mixture was then stirred for a further minutes at 0 and concentrated to its original volume in vacuo. The residue was then partitioned between ethyl/ acetate and hydrochloric acid, washed with hydrochloric acid, water and concentrated sodium chloride solution during which in order to prevent the formation of a precipitate some methanol was added from time to time, or the mixture was subjected to slight heating. The

Example 7 Example 8 N formyl-(N -ZyD-Dab-(N -Palm)-L Dab one The above was prepared from l- -(N lr almyh-Dao OCH .HCl and N-formyl-(N'-Z)-D-DabOH using dicyolohexylcarbodiimide in dimethylformamide in accordance with the method set forth in Example 1 above. Upon. crystallization from ethyl acetate it melted at 164- 166.

Nf0rmyl- (N -Z -D-Dab-( N -Palm -L-Dab NHN H The above was prepared from above-mentioned ester by using hydrazine hydrate according to the method of Example 3. Upon crystallization from methanol, it melted at N L-206.

H-D-Dab-(N -Palm)-L-Dab-NHNH .2HBr: 10 g. of N -forrnyl-(N*-Z)-D-Dab-(N"-Palm -L-Dab-NHNl-l was covered with 120 ml. of 33% hydrogen bromide/glacial acetic acid and'with the exclusion of moisture shaken for 2 hours. The so-formed emulsion was treated with 500 ml. of absolute ether and the precipitated product per mitted to stand for 2 hours in 50 ml. of water at 20 order to effect deformylatiou. The end product was then isolated by lyophilisation and recrystallized from methanol/ethyl acetate. It melted at 215-218 (dec.).

Example 9 ;N-Z-(N"-Z)-D Dab-(N"-Z)-D Dab (N Palm) L- Dab-O CH 8.4 g. of H-(N -Palm)-L-DaJb'OCH .HCl was dissolved in ml. of dimethylformamide and vigorously stirred for 15 minutes with 3.2 m1. of triethylamine. The mixture was then filtered with suction and 13 g. of N-Z-(N--Z)-D-Daib-(N'-Z)-D-Dab-OH dissolved in the filtrate, and the so-formed mixture then treated at 0 with 4.3 g. of dicyclohexylcarbodiimide. After 24 hours, the dicyclohexylurea which had been suction.

formed was filtered off with suction, and the filtrate treated with 20 g. of sodium chloride in 1 liter of 0.1 N hydrochloric acid. The resulting mixture was then filtered with suction, the precipitate mixed with dimethylformamide with 0.1 N ammonia and reprecipitated with the addition of sodium chloride. The precipitate was dried in vacuo at 70 andcrystallized from methanol, yielding N"-Z-(N-Z)-D-Dab-(N -Z)-D-Da b-(N-Palm) L Dab- OCl-l melting at 178-180".

Dab-(N -Pa lm)-L-Dab-OCH was dissolved in 100 m1. of,

dimethylformamide and 14 ml. of 100% hydrazine hydrate with warming, and then allowed to stand for 24 hours at 20. It was then triturated with 200 ml. of ethanol, filtered with suction and the precipitate washed with ethanol and dried, yielding N-Z-(N"-Z)-D-Dab- (N-Z)-D-Dab (N"-Palm)-L-Dab-NHNH melting at 222-224".

H-D-Dab-D-Dab- NPal-m -l'.-Dab-NHNH .3HBr: g. of NZ-(N-Z)-D-Dab-(N-Z)-D-Dab-(N-Palm) L- Dab-NHNH was covered with 75 m1. of 33% hydrogen bromide/glacial acetic acid and shaken for 2 hours with the exclusion of moisture. The suspension was then mixed with 300 ml. of absolute ether, the supernatant mother-liquor decanted oil? and the residue washed several times with fresh absolute ether. It was then recrystallized from methanol/ether yielding H-D-Dab-D Dab-(N -Palm)-L-Dab-NHNH .3HBr which showed a rotation of [m] ='7.8 (c.=1 in water).

Example 10 N.-forrnyl-(N" -Z)-L-Dab-(N"-Z)-D-Dab (N -Z) D- Dab-(N-Palm)-L-Dab-OCH 9.1 g. of N"-for1nyl-(N" Z)-L-Dab-(N7-Z)-D-DahNHNI-I was dissolved with vigorous stirring at 0 in 100 ml. of glacial acetic acid, 50 ml. of water, 100 ml. of ethyl acetate and 12.7 ml. of 3 N hydrochloric acid. A solution of 1.32 g. of sodium nitrite in ml. of water was then slowly added thereto in a dropwise manner at l0. Following 15 minutes the mixture was extracted with ethyl acetate at 0, washed with water and 1 N sodium bicarbonate, and the solution dried over sodium sulfate at 0. The so-obtained azide solution wasthen treated with a solution of 11 g. of H- (N"'-Z)-D-Dab-(N"'-Palm)-L-Dab-OCH in 65 m1. of dimethylformamide, the resulting mixture permitted to stand at 0 for hours and then at 20 for 6 hours, after which it was concentrated in vacuo and mixed with 400 ml. of ether yielding as a precipitate, N formyl-(N-Z)- L-Dab-(N' -Z) -D-Dab-(N"-Z)-D-Dab-(N-Pa.lm)-L -Dab- 0CH which, after being filtered off with suction, washed with ether and dried in vacuo at 60, melted at 222-224".

Z) L Dab-(N -Z)-D-Dab-(N -Z)-D-Dab-(N -Palm)-L- Dab-OCH was dissolved in 90 ml. of dimethylformamide and 11ml. of 100% hydrazine hydrate with warming. After 20 hours, the solution was mixed with 200 ml. of water, filtered with suction and the precipitate washed with water "and dried in vacuo at 80, yielding N-formyl- (N Z) L Dab (N Z)-D-Dab-(N -Z)-D-Dab-(N Palm)-L-Dab-NHNH melting at 240-242".

H L Dab-D-Dab-D-Dab-(N"-Palm)-L-Dab-NHNH 4HBr: 8 g. of N-formyl-(N"-Z)-L-Dab-(N"'Z)-D-Dab- (N -Z)-D-Dab-(N-Palm)-L-Dab-NHNH Was covered with 80 ml. of 33% hydrogen bromide/glacial acetic acid and shaken for 2 hours with the exclusion of water. The resulting suspension was mixed with 300 m1. of absolute ether and the supernatant mother-liquor filtered off with The residue was treated several times with fresh ether and then taken up in 80 ml. of water. Any remaining ether was removed by evacuation, and the mixture then permitted to stand for 2 hours at 20. The aqueous solution was then lyophilised and the residue recrystallized from methanol/ether. It was then dissolved in methanol, neutralized with pyridine to a pH of 7, and the solution precipitated out in alcohol. After drying, the

precipitate was dissolved in 50 ml. of water and lyo- Example 11 N-( l0-undecenoyl -L-Lys-LLys-OC H .2HCl: 28 g. of H-(N-phthaloyl)-L-Lys-OC H .HCl was dissolved in 150 ml. of dimethylformamide by slight warming. After cooling, it was treated with 12 ml. of triethylamine and from triethylamine hydrochloride filtered oil with suction. 35 g. of N-(IO-undecenoyl)-(N-phthaloyl)-L-Lys-Ol-I was then added to the filtrate and dissolved by slight warming. The reaction mixture was then cooled to between 0 and 5 and 17.5 geof dicyclohexylcarbodiimide in ml. of dimethylforrnamide added thereto. The resulting mixture was then placed in a refrigerator. After 24 hours the urea formed was filtered off, the solution mixed with ice water, the precipitated protected dipeptide dissolved in ethyl acetate, washed with l N sodium bicarbonate, 01 N hydrochloric acid and water. After evaporation, the residue was recrystallized from ethanol/ Water yielding N-( lO-undecenoyl)-(N-phthaloyl)-L-l ys- (N phthaloyl) L Lys OC H melting at -132"; [a] =7.4 (c.=2.54 in ethanol).

In order to split off the protecting groups, 7.3 g. of the above described dipeptide was'heaied under reflux for l hour in 100 ml. of ethanol and? ml. of water with the addition of l g. of hydrazine hydrate. After standing for some time at room temperature, it was treated with 2 ml. of concentrated hydrochloric acid, shaken thoroughly and with suction filtered free of the phthaloyl hydrazide which had been formed. Concentration of the ethanolic mother-liquor and filtration resulted in separation of still more phthaloyl hydrazide. The filtrate was then concentrated, and precipitated with acetone and petroleum ether yielding N (IO-undeenoyl)-L-Lys-L-Lys-OC H .2HCl which, after being recrystallized twice from ethanol, melted at 241-243"; [a] =31i2 (c.=1.6 in water). I

Example 12 N,N-di-Z-L-Lys- N-Z -L-Lys-cetylamide: A solution of 35.4 g. of N,N di-Z-L-Lys-(hV-Z)-L-Lys-OH (J. Chem. Soc., 1953, 475) in ml. of tetrahydrofuran was treated at 2 with stirring with 8.6 g. of carbonyl-diimidazole. After 30 minutes 12.7 g. of cetylamine was added thereto, and the mixture permitted to stand overnight at room temperature. The precipitated substance was then filtered off with suction and washed, yielding 19.8 g. of product. The filtrate in turn was concentrated in vacuo taken up in ethyl acetate, washed and dried. Following evaporation and recrystallization from chloroform/petroleum ether it yielded 5.2 g. of further product. All together there was obtained 25.0 g. of N,N -di-Z-L-Lys- (N-Z)-L-Lys-cetylamide melting at 147-152"; [od =63 (c.=2.0 dimethylforamide).

H-L-Lys-L-Lys-cetylamide.3HC1: 28 g. of N,N-di-Z- L-Lys-(N -Z)-L-Lys-cetylamide was decarbobenzoxylated by treatment with 50 ml. of 33% hydrogen bromide in glacial acetic acid for 2 hours. After precipitation with ether and distillation with 50 ml. of methanol, the residue was dissolved in a minimum of water and added to 80 g. of Amberlite IRA 410. It was then eluted with water, filtered through charcoal, treated with 30 ml. of 3 N hydrochloric acid and concentrated to 40 ml. at 50 and 60 mm./ Hg. After precipitation with acetone, it was filtered with suction, dried over phosphorus pentoxide, taken up in hot ethanol, filtered through charcoal and precipitated via the slow addition of acetone, yielding 6.3 g. of. H-L-Lys-L-Lys-cetylamide.3HCl, melting at 230-250 (dec.); [a] =|6.6 (c.=2.0 in methanol).

iii Example 13 N Z (N phthaloyl)-L-Lys-(N-phthaloyl)-L-Lys- OC H 10.5 g. (31 mmole) of hF-phthaloyl-L-Lys- OC H HCl was dissolved in 70 ml. of dimethylformamidc, treated with 4.4 ml. of triethylamine and filtered. The filtrate was at once added to a solution of 12.6 g. (31 mmoles) of N l-(N phthaloyl)-L-Lys-OH in 150 m1. of absolute tctrahydrofuran, and then 6.4 g. (31 mmoles) of dicyclohexylcarbodiimide was added to the mixture.

1., 46, 582 (1950)] in 150 ml. of 1.5 N potassium hydroxide and 1 00 ml. of tetrahydroturan was treated droptwise at 25 with 30 g. (0.11 mole) of palmitoyl chloride. Following the addition, the mixture was stirred [for a further 40 minutes with occasional addition of 2 N potassium hydroxide in order to maintain the medium alkaline. was then acidified (Congo red) with hydrochloric 'acid and the tetrahydrofuran evaporated in vacuo. To the residue there was added 500 ml. of ethyl acetate and a small After being permitted to stand for 16 hours at 2-4", the amount olfhoonoentmted z g g g 23 h g lgfi 'dicyclohexylurea formed was filtered OH and the filtrate a ase was separa e 0 an e remain l' evaporated in vacuo. The residue was taken up in ethyl me addmon of Small amount of methanol Washed l acetate and 1 N hydrochloric acid, filtered free of further f f l water gi g ff g sodium urea, washed with 1 N hydrochloric acid, water, 10% sor, g r i e 31 dium bicarbonate saturated sodium chloride solution, irate vacuo P 1 l e i lz mm dried over sodium sulfate and evaporated in vacuo yieldmethane Water yielded N g' f ing N Z (N -phthaIoyI)-L-Lys-(N-phth-aloyl)-L- OH melting at 91-96 [a] .8 (c.=1.0 111 etha- OC H which, upon recrystallization from ethyl acetate/ petroleum ether, melt d at 116421"; 11.8 The above described procedure was used to prepare the 5 i h l) N-acylated N"-Z-arnino acids listed inthe ttollowing talble:

' Cf. Example M.P Cryst.l'rom 1 N -Pa1rn-(N*-Z)-LLys-0II 101-102 (Hydrogen Chloride/water). 4a.. N' Palrn-(N-Z)-D-Lys-OH 101-102" Do. 41L. N -Palm-(N'-Z)DL-Lys-OH 134-135" (Ethyl Acetate/Petroleum Ether). 4e N-Steuroy1-(N-Z)-L-Lys-OH 95-97 (Ethyl Acetate/Petroleum Ether). 4h N- Lauroyl-(N -Z)-L-LyS-OH 102-104 (Ether/Petroleum Ether). 4i N -Palm-(Nv-D-L-Dab-OH 94-96 (Methanol/water). 4mm, N -Capryl-(N D-L-Lys-OH 102-106D (EthylAcetate/Petroleum Ether).

. cording to the method already described above, yielding H (N phthaloyl) L Lys-(N'phthaloyD-Llys- "OC l-l HBr which upon recrystallization from chloroform and a little ethanol/ ether melted at 230-235 N Oleoyl (N phthaloyl) L Lys-(N-phthaloy-l)- L-Lys-OC H5: 12.9 g. mmoles) of the above-obtained hydrobromide was liberated in 35 ml. of chloroform with 3 ml. of triethylamine, then treated with 100 ml. of tetrahydrofuran at 20, filtered and treated with a tfurther 3 ml. of tniethylamine. Then, with good stirring under a nitrogen atmosphere and at 5.6 g. (18.6 moles) of oleoyl chloride was added thereto dropwise, the resu-Iting mixture then stirred for a durther /2 hour at 6 and then evaporated in vacuo. The residue was taken up in warm ethyl acetate/1 N hydrochloric acid, separated in a preheated separating funnel and rapidly washed with warm water and concentrated sodium chloride solution, dried and concentrated in vac-no. The residue was then crystallized ttrom ethyl acetate/petroleum ether and ethanol/ water yielding 12 g. of 'N-oleoyl-(N-phthaloyl)-L- Lys- (N -phthaloyl)-L-Lys-OC H melting at 1 18- 125; [a] =9.7 (1.0 in ethanol).

N oleoyl L Lys-L-Ly-s-OC H HO1: In order to split off the phthaloy'l protecting groups 9.7 g. (11.7 mrnoles) of the above obtained product was dissolved in 100 ml. of ethanol and following the addition under a nitrogen atmosphere of 3.5 ml. of 6.72 N hydrazine hydrate, heated for one. hour under reflux. Atfter cooling, it was treated with 8.0 m1. of 3.2 N hydrogen chloride in ethanol, held at room temperature overnight and with suction filtered free of phthaloyl hydrazide. The filtrate was concentrated in vacuo and after further filtration Olf phthaloyl hydrazide, twice recrystallized from ethanoi/ ether, yielding 3.3 g. of N oleoyl L Lys =L Lys- OC H .2HC1 which decomposed above 230 without melting; [a] =-17.7 (c.=1.0 in ethanol).

The starting materials used in the foregoing examples were prepared as tfollows:

N-Palm-(N -Z)-L-Orn-OH (cf. {Example 4f): A solution of 265 g. (0.1 111. 1%) of N"-Z-LOrn-QH [Bioohem.

The amino acid esters were prepared either according to the standard procedure of C-urtius and Goebel, I. prakt. Chemie [2], 3.7, 150 (1888), with alcohol/hydrogen chloride or by means of alcohol and thionyl chloride according to the procedure of Brenner and Huber, Hel-v. Ohimica Acta, 36, 1109 (1953).

H-(N -Pahn)-L-Lys-OCH (of. Example 41:): 24 g. of H-L-Lys-OH.HC1 was dissolved in 1500 ml. of water and heated under reflux for 2 hours with g. of basic copper carbonate. tit was then filtered and treated at 0 with 40 g. of palmitoyl chloride and sufficient 2 N sodium hydroxide in order to maintain the reaction mixture basic. After 2 hours, the precipitated copper complex was filtered oil, washed with water, acetone and ether and dried. The powder was then suspended in 500 ml. 01f methanol and ml. of water, acidified with concentrated hydrochloric acid and treated tor /2 hour at with hydrogen sulphide. The copper sulphide was then filtered off and the filtrate adjusted to pH 7 with sodium hydroxide. After standing for 4 hours at room temperature, the mixture was filtered and the residue crystallized from glacial acetic acid/ether yielding -H(NPalm)-L-Lys-OH melting at 240.

30 ml. of anhydrous methanol was treated at -10 with 2.7 ml. of thionyl chloride. 14 g. of H-(N-Palm)-L-'Lys- 0H was then added to the mixture. Ariter 3 hours the clear solution was evaporated in vacuo at 45 the residue dissolved in methonal and crystallize-d by the addition of ether. The crystals were filtered 01? with suction, washed with ether and dried, yielding H-QN PaIrM-L- Lys-OCI-l .HC1 melting at 142143.

li-I-(N -Palm)-L-Dab-OCH (cf. Example 8): 41 g. of H-L-Dab-OHHCI and 35 g. of basic copper carbonate in 300 ml. of water were heated with stirring for 2% hours under reflux. Following filtration with suction, the filtrate was treated with 60 g. of sodium bicarbonate and, while stirring at 20, treated dropwise with a solution of 73 g. of palmitoyl chloride in 200 ml. of acetone. The mixture was then stirred for 16 hours at 20, the copper complex filtered off with suction, washed with a large amount of water and acetone, dissolved in 200 ml. of water and 500 ml. of methanol with the addition of concentrated hydrochloric acid, and treated with hydrogen sulphide until no more copper sulphide separated out.

mixed at 10, treated with 36 g. of H-(N"-Palm)-L- Dab-OH and warmed under reflux at 50 for 6 hours with the exclusion of moisture. The solution was then concentrated in vacuo at 50 and recrystallized from methanol/ ether yielding H-(N'Palm)-L-Dab-OCH .HCI melting at 144-146.

N-formyl-(N -Z)-D-Dab-OH (cf. Example 8): Cf. Helv. Chim. Acta, 43, 279 (1959).

N-Z-(N-Z)-D-Dab-OH (cf. Example 9): Cf. Chem.

.Listy, 47, 427 (1953).

D-Dab-OH by use of carbodiimide according to the process of Example 1, N-Z-(N'-Z)-D-Dab-(N"-Z)-D-Dab- OCH melting at 122-124 was obtained. The corre sponding dipeptide acid was obtained by saponification using the calculated amount of alkali. Said acid melted at 163-165".

N"-formyl-(N"-Z)-L-Dab-(N" Z) L D Dab OCH (cf. Example 10): This was prepared from the components in accordance with the procedure in Example 1; M.P. 138-441".

N-formyl-(N -Z)-L-Dab-(N'-Z)-D-Dab NHNH (cf. Example 10): This was prepared from the corresponding este'rusing hydrazine hydrate in accordance with the procedure set forth in Example 3; M.P. 196200.

H-(N -Z)-D-Dab-(N -Palm)-L-Dab-OCH (cf. Example 10): This was prepared from N"-formyl-(N -Z)-D- Dab- (N'-Pa1m)-L-Dab-OCH and methanolic hydrochloric acid at room temperature.

H-(N-phthaloyl)-L-Lys-OCH .HCl (cf. Example 11): 72 g. of I-I-(N-phthaloyl)-L-Lys-OH (Rec. Trav. Chim. Pays-Bas, 79, 688 [1960]) was covered with 1500 ml. of hydrogen chloride/ethanol and refluxed at 60 for 72 hours with the exclusion of moisture. It was then concentrated and precipitated with ether. The precipitate was recrystallized from ethanol/ ether, yielding the hydrochloride of H-(N-phthaloyl)-L-Lys-OC H which melted at 175-177", [u] =+12.3 (c.=2 in water).

N-(10-undecenoyl)-(N-phtl1aloyl)-L-Lys-OH (cf. Example 11): For the preparation of N-(10-undecenoyl)- (Npht-haloyl)-L-Lys-OH, 3.3 g. of the corresponding ester hydrochloride was dissolved in 30 ml. of dimethylformamide, 1.5 ml. of triethylamine added thereto and the mixture with suction filtered free of the so-formed triethylamine hydrochloride. Then 1.9 g. of 10-undecenic acid was added thereto, the resulting mixture thoroughly cooled and treated with a solution of 2.2 g. of dicyclohexylcarbodiimide and 10 ml. of dimethylformamide. After 20 hours the urea formed was filtered ofl and the filtrate precipitated with ice/sodium chloride solution. The precipitate was then taken up in ethyl acetate, washed with sodium bicarbonate, hydrochloric acid and water and dried. Recrystallization from ethyl acetate/ petroleum ether yielded purified N-( l-undecenoyl)-(N- phthaloyl)-L-Lys-OC H 21 g. of this latter substance was dissolved in 250 ml. of acetone. 50 ml. of water and 25 ml. of concentrated hydrochloric acid were added thereto, and the resulting mixture boiled under reflux for 3 /2 hours. It was then concentrated in vacuo at 40, extracted 3 times with ethyl acetate and recrystallized from ethyl acetate/petroleum ether yielding N-(-undecenoyl)-(N-phthaloyl)-L-LysOH melting at 114-116.

N-Z-(N'-phthaloyl)-L-Lys-OH (cf. Example 13): g. (0.048 mole) of N -PhthalOyl-L-Lys-OH.HC1 was dissolved in 150 ml. of water and 100 ml. of acetone and then stirred at 4-5 together with 6 g. of magnesium oxide, following which during the course of 45 minutes 9.0 g. (0.053 mole) of carbobenzoxy chloride in 30 ml. of acetone was added to the mixture dropwise. After stirring for a further 4 hours at 510 the mixture was acidified, concentrated in vacuo to /2 its volume and extracted with ethyl acetate. extracted with 5% potassium bicarbonate and theextracts after acidification were further extracted with ethyl acetate. Drying and concentration in vacuo yielded 14.5 g. of N"-Z-(N-phthaloyl)-L-Lys-OH in the form of a glasslike mass which did not crystallize.

Example 14 cient amount of the same substance was added to the third flask to bring about a final concentration of 0.01%. The flavor and odor of the grape juice was unchanged by these additions. The flasks were closed with a plug of cotton wool and allowed to stand at room temperature. While the content of the first flask strongly fermented after 4 days, fermentation first began in the 0.001% addition flask after about 6days and the flask with the 0.01% addition showed no appearance of fermentation ofany sort after 14 days and was no different in flavor and odor from freshly strained grape juice.

Analogous resultslwere obtained with apple and carrot juice.

Example 15 3 portions of g. each of) minced meat were wrapped in sterile gauze. In the case of the first portion, the gauze was moistened with sterile water. the second portion, the gauze was moistened with a 0.1% solution of N"-Palm-L-Lys-L-lysine ethyl ester dihydrochloride, and in the case of the third portion, the gauze was moistened with a 1% solution of the aforementioned substance (each of the solutions was prepared with sterile distilled water). While the control portion was already unsightly after 2 days at room temperature and possessed an odor' of tainted meat, the other 2 remaining portions retained'their original color for 2 further days at room temperature, and the odor of tainted meat was practically absent.

In a similar way pork slices and cut ham were tested, but these were also in addition packed in plastic foil in order to avoid drying out. A favorable effect of the additive was also observed in these cases.

Example 16 Six slices of black bread (Westphalian rye bread or -10 days, the sample which had been sprayed with tap water was completely deteriorated while the slices'which had been treated with the 0.1% solution showed only slight local cultures of mold and the slices of bread which had been treated with the 1% solution showed no preceptible change at all.

Example 17 r 28 oranges were each punctured ten times with a needle in order to facilitate the penetration of microorganisms.

This was then thoroughly v In the case of anemone Subsequently 7 oranges were each immersed 1 minute in one of the three following solutions:

i lnys lat llnioh l M Hutch 3 (ontrol hatches with addition of l llll. of distilled water and (Li ml. of yeast suspension.

Legend: -=no fermentation; +=woak fermentation; +I-=stron fermentation; +++=vory strong fermentation.

After draining, the 21 oranges were evenly sprayed with 10 a suspension of spores which originated from an orange Example 19 which had become moldy. The oranges were then Wrapped up in groups in paper which had been moistened 50 ml. flasks each contaming 19 ml. of freshly strained with Water The remaining 7 Oranges were treated with orange uice were each treated with 1 ml. of a sufficiently solution 1 above, sprayed and wrapped up in paper which in place of Water had been moistened with solution 3 above. The fruits were then laid out at room temperature and prevented from drying out by covering with plastic foil. The results are given in the following table.

concentrated solution of a preservative indicated in the table below, so that the end concentration indicated in the table was obtained. The flasks were then stored at room temperature (about and the mold formation evaluated after the third, fifth, seventh and ninth days.

The numbers indicate the number of oranges on which mold attack could be observed,

and the signs indicate the degree oi attack as follows:

+=Slight mould. ++=A large amount of mould.

I Example 18 End Concentration of 50 ml. fiasks, each containing 19 ml. of white grape P D PreservotivoinPereent juice, were each treated with 1 ml. of a concentrated solureservaflve Agent ays tion of a preservative indicated in the table below in a 0.05 0.01 0.005 0001 quantity sufficient so that the end concentration shown in the table below was obtained. In order to facilitate g g l fi ys L- v fermentation, 0.1 m1. of a 10% suspension of a bakers I Y we on 7 I I I I. yeast in water (that is 10 mg. of yeast per batch) was 9 added to each of the flasks. The amount of fermenta- Nmpalmlkomrlrmgmmo 3 tron was evaluated after storage at room temperature methylesm dlhymochloride i (about 20) after the first, second, third and fourth days. 9

5O N -PaIm-L-Orn-L-Qruithine 3 End mncentmtion of the methyl ester dihydrochloride. g i preservative agent in percent Prcservotive Agent Days 9 1 N -Pa.lm-L-LysL-Lysine ethyl/ 3 I [L005 0-001 ester dihydrochloride. 5

ri y roci ori e. u 4 7 I N -PalIIi-L-t0r1-%a5glnln6 i+ 9 methy es er i y m- 0 N-Pa1m-L-Lys-L 1eucine 3 chloride. 3 m 1 t d hi 1 5 4 me y es or by roe on 0 7 i i+ fi i+ N Palm-L-Orn-L-ornithine 1 9 nfithyillester dihydrog 1 I 6. I 1 4 ii ii Days 1st Batch 2d Batch 6f N -Pahn-L-LyS-L-Lysine 1 I ethyl/ester dihydroehlo- 2 2 control batches wlth addition of 1 3 ride. 3 ml. of distilled water. g i+ ii+ 9 L-Lys-L-Lys-hexadecyl- 1 amide trihydrochloride. 2 h

3 i 40 Legend: 4 1:4. g -=No mold formation.

+=Small amount of mold (up to about 10%v of the surface N -Palnu-L-LysL-leueine 1 eovered) methyl/ester hydrochlo- 2 ++=La5)ge amount of mold formation (about of the surface ti o. 3 covers q 4 i-l- +++=Very large amount vof mold formation of the sur- 7 face covered).

messes 21 Example 20 50 ml. of vinegar and 100 ml. of peanut oil, together with 750 mg. of N -Palm-L-Lys-L-lysine ethyl ester dihydrochloride, as emulsifying agent, were emulsified for one minute at a speed of 10,000 r.p.m. A control experiment,

where the emulsifying agent was omitted, was run simultaneously. Whereas the control mixture separated into two layers after about one hour, the mixture containing the emulsifying agent remained as an emulsion for more and acid addition salts thereof; wherein n, and n are each a whole integer from to 1; X is selected from the group consisting of hydroxy, alkoxy, amino, alkylamino, dialkylamino, and hydrazino; each R is the residue of an a-amino acid devoid of its amino and carboxyl group, at least one R being the residue of a diaminocarboxylic acid; and the molecule contains at least one long-chain aliphatic group selected from the group consisting of a long-chain aliphatic acyl substituent on a free amino group and a long-chain aliphatic substituent comprehended by X whenX is selected from the group consisting of alkylamino and dial kylamino.

2. Acompound as in claim 1 wherein in and n are each 0.

3. A compound as in claim 1 wherein at least one longchain aliphatic group contains from to carbon atoms.

4. A compound'selected from the group consisting of (N -palmitoyl-L-lysyl)-L-lysine, alkyl esters thereof, amides thereof and hydrazides thereof, and acid addition salts of each.

5. A compound selected from the group consisting of L-lysyl-(N-palmitoyl-L-lysine), alkyl esters thereof, am-

ides thereof and hydrazides thereof, and acid addition '(N"-palmitoyl-L-arginyl)-L-ornithine, alkyl esters thereof,

amides thereof and hydrazides thereof, and acid addi tion salts of each.

10. A compound selected from the group consisting of (N-palmitoyl-L-arginyl)-L-arginine, alkyl ester thereof, amides thereof and hydrazides thereof, and acid addition salts of each.

11. A compound of the formula wherein R1 is selected from the group consisting of lower alkylene and -(CH NH-C(=NH); Y is selected from the group consisting of hydroxy and alkoxy; one of the 'two amino groups is substituted by a long-chain acyl group and the other amino group is selected from the group consisting of primary amino and amino protected with'a removable protecting group.

12. A compound of the formula wherein R is selected from the group consisting of lower alkylene and (CH NHC(=NH); Y is selected from the group consisting of hydroxy and alkoxy; one of R and R is a long-chain acyl group and the other is hydrogen.

13. (N-palmitoyl-L-lysyl)-L-lysine ethyl ester dihydrochloride.

14. (N -palmitoyl-L-lysyl)-L-lysine amide dihydrochloride.

15. (N"-palmitoyl-L-ornithyl)-L-arginine methyl ester dihydrochloride.

References Cited by the Examiner UNITED STATES PATENTS 2,592,454 4/1952 Mowat et al 260-l12 2,668,115 2/1954 Wolf 99-154 2,668,116 2/1954 Wolf 99--l54 2,843,525 7/1958 Robinson et al. 260-1l2.5 2,850,491 9/1958 Brenner 260-412 FOREIGN PATENTS Ad. 71,405 7/ 1959 France.

OTHER REFERENCES Boissonnas et al., Chem. Abs. volume 55, No. 14, p. 13325-26.

I Bondi et al., Biochemische Zeitschrift, volume 23, 1910, pp. 499-509.

LEWIS GO'ITS, Primary Examiner.

HYMAN LORD, Examiner.

P. A. STITH, Assistant Examiner.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTIGN Patent No, 3,265,682 August 9 1966 Urs Gloor et aln It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 2, line 18, for "undecanoyl" read undecenoyl line 33, for "diaminobutyryl-L" read M diaminobutyryl)] L column 5, line 29, for "benzyloxycarbonyl" read benzyloxycarbonyl-L- column 5, line 30, for "benzyloyl" read benzyloxy line 52, for "polmitoyl" read M palmitoyl column 6, line 21, for "N read [N line 23, for "orginyl" read arginyl column 8, line 8, for "N-(N" read H-[N line 39, for "N-Palm" read N -Palm column 14, line 59-, for "=6.,3" read =-63 column 15, line 44, for "20" read 20 column 16, line 17, for "+.8" read +4.8 column 19, line 67, second Table, sixth column, line 15, for read Signed and sealed this 22nd day of August 1967.

(SEAL) Attest:

ERNEST W. SWIDER EDWARD JG BRENNER Attesting Officer Commissioner of Patents 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF COMPOUNDS OF THE FORMULA: 